Fig. 1.

Gene targeting at the LHβ locus. (A) A phosphoglycerate kinase-neomycin expression cassette was engineered to contain a 5′ loxP sequence and on the 3′ side another loxP sequence followed by five stop codons. This cassette was inserted into exon 2 of the mouse LHβ gene in ES cells by homologous recombination (arrowhead). The floxed phosphoglycerate kinase-neomycin cassette was flanked by 6.3 kb of 5′ and 6 kb of 3′ LHβ gene sequences originally cloned from a 129S6/SvEv mouse genomic library. (B) Southern blot shows WT and mutant alleles identified by their size (7.3 and. 3.4 kb, respectively) to distinguish the genotypes of mice. (C) Dual immunofluorescence of pituitary sections shows that LHβ-specific staining (labeled green) was absent in a pituitary section from the null mouse (KO) compared with that in control (WT). FSHβ-specific staining (in red) is seen in both control and null mice. (D) Immunoblot of pituitary proteins shows the presence of LHβ-reactive band only in control pituitary extracts (+/+ and +/–) but not in that of the null (–/–) mice, thus confirming that we engineered a null mutation at the LHβ locus. Because heterodimer assembly confers biological activity to glycoprotein hormones, absence of LHβ-subunit leads to LH deficiency in these null mice.